(1) Field of the Invention
This invention relates to preparative electrofocusing in flat bed granulated polysaccharide gels.
(2) Descriptions of the Prior Art
The technique of electrofocusing is used to separate protein mixtures on the basis of differences in net charge in a stable pH gradient. If an ampholyte mixture is distributed along a support between two electrodes and a direct current applied through the ampholyte mixture at equilibrium the electro-migration of ampholytes towards the electrodes and the opposing diffusion flow are balanced with the various ampholyte components distributed in the support from anode to cathode in order of increasing pI. The concentrations of each component will be distributed approximately in gaussian fashion about their respective pIs.
The technique was discussed by Svensson (Acta Chem. Scand. 15 (1961) 325-341; Acta Chem. Scand. 16 (1962) 456-466). It was suggested by Svensson that all ampholytes with pI-pK, values bigger than 2.5 units (which includes all neutral amino acids) were useless as carrier reagents and those with pI-pK, values between 1.5 and 2.5 units were poorer carriers. Subsequently there were developed a series of synthetic polyamino-polycarboxylic acids sold under the trade mark "Ampholines" which had advantages as carrier ampholytes in comparison with known ampholytes at the time. However these ampholytes are very costly and have a tendency to strongly complex with both acidic and basic molecules modifying their pattern obtained. Also they tend to bind to stains commonly used for the localisation of protein zones giving rise to staining difficulties in the detection of minor protein components.
Chrambach et.al. (Anal. Biochem. 74 145 (1976) have used systems containing polyacrylamide gel rods as the supporting medium and more common buffers as ampholytes. However, these studies have been directed primarily at microanalytical applications and are of limited value for preparative separations.
It is an object of this invention to provide an electrofocusing system which can be used for preparative electrofocusing of protein mixtures. The method described in this invention permits large sample loadings of mixtures of proteins and other biological substances and the focused zones can be easily recovered in high yield without significant loss of biological activity.